Prenatal phenotypes and pregnancy outcomes of fetuses with 16p11.2 microdeletion/microduplication

Background Chromosomal 16p11.2 deletions and duplications are genomic disorders which are characterized by neurobehavioral abnormalities, obesity, congenital abnormalities. However, the prenatal phenotypes associated with 16p11.2 copy number variations (CNVs) have not been well characterized. This study aimed to provide an elaborate summary of intrauterine phenotypic features for these genomic disorders. Methods Twenty prenatal amniotic fluid samples diagnosed with 16p11.2 microdeletions/microduplications were obtained from pregnant women who opted for invasive prenatal testing. Karyotypic analysis and chromosomal microarray analysis (CMA) were performed in parallel. The pregnancy outcomes and health conditions of all cases after birth were followed up. Meanwhile, we made a pooled analysis of the prenatal phenotypes in the published cases carrying 16p11.2 CNVs. Results 20 fetuses (20/20,884, 0.10%) with 16p11.2 CNVs were identified: five had 16p11.2 BP2-BP3 deletions, 10 had 16p11.2 BP4-BP5 deletions and five had 16p11.2 BP4-BP5 duplications. Abnormal ultrasound findings were recorded in ten fetuses with 16p11.2 deletions, with various degrees of intrauterine phenotypic features observed. No ultrasound abnormalities were observed in any of the 16p11.2 duplications cases during the pregnancy period. Eleven cases with 16p11.2 deletions terminated their pregnancies. For 16p11.2 duplications, four cases gave birth to healthy neonates except for one case that was lost to follow-up. Conclusions Diverse prenatal phenotypes, ranging from normal to abnormal, were observed in cases with 16p11.2 CNVs. For 16p11.2 BP4-BP5 deletions, abnormalities of the vertebral column or ribs and thickened nuchal translucency were the most common structural and non-structural abnormalities, respectively. 16p11.2 BP2-BP3 deletions might be closely associated with fetal growth restriction and single umbilical artery. No characteristic ultrasound findings for 16p11.2 duplications have been observed to date. Given the variable expressivity and incomplete penetrance of 16p11.2 CNVs, long-term follow-up after birth should be conducted for these cases.

Currently, most studies involving 16p11.2CNVs are identified through postnatal evaluation.However, the intrauterine phenotypic features associated with 16p11.2CNVs are not well described, which poses a challenge for genetic counseling and prenatal management for these carriers.To enhance the prenatal knowledge on 16p11.2CNVs, we present the clinical and molecular findings of 20 cases with 16p11.2deletions and duplications in the pregnant women who opted for amniocentesis.Additionally, we systematically reviewed the prenatal phenotypes associated with such chromosomal disorders.

Clinical data
This retrospective study was performed from October 2018 to November 2023 and enrolled 20 cases with 16p11.2microdeletions and microduplications selected from 20,884 pregnant women.These women were referred to the First Hospital of Jilin University for invasive diagnostic testing via amniocentesis.The main indications for prenatal diagnosis included non-invasive prenatal testing (NIPT) for aneuploidy, maternal serum screening results for aneuploidy, ultrasound anomalies (structural or non-structural), parental chromosomal abnormalities, abnormal childbearing history, advanced maternal age, and voluntary request.All pregnancy women accepted routine prenatal ultrasound examinations during the gestation period, and abnormal ultrasound findings were included in the indications for prenatal diagnosis.All couples denied consanguineous marriage, and the pregnant women denied any exposure to teratogenic agents, irradiation, or infectious diseases during this pregnancy in question.After acquiring genetic testing results, all prospective parents received prenatal genetic counselling, and blood samples were collected with informed consent.The study protocol was approved by the Ethics Committee of the First Hospital of Jilin University (No. 2021 − 706), and written informed consent was obtained from all the couples.

Cytogenetic analysis
Pregnant women underwent amniocentesis for karyotyping analysis with written informed consent.30 mL of amniotic fluid cells were collected.Routine cytogenetic analysis was performed using G-band metaphases at 400-500 banding resolution, which were prepared from 20 mL of cultured amniotic fluid cells in accordance with standard protocols in our lab.Twenty metaphases were analyzed for all samples according to the International System for Human Cytogenetic Nomenclature 2016.

Selection of prenatally detected 16p11.2 microdeletions and microduplications
In order to summarize the prenatal phenotypes of 16p11.2deletions and duplications in the published reports, we launched a literature review for identifying relevant articles from inception to 2023.Criteria for the selection were defined as English and Chinese languages, 16p11.2deletions and duplications, CNVs and prenatal phenotypes.The English language database PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) and the Chinese language databases (Wanfang Data and China National Knowledge Infrastructure) were searched.CNVs with chromosome coordinates for all reviewed cases were required to be provided.A string of the following terms and their synonyms was utilized: 16p11.2deletion/loss, 16p11.2duplication/gain, prenatal diagnosis, chromosomal microarray analysis, and ultrasound findings/intrauterine phenotype.The combination of subject words and free words was also used for the search.The prenatal phenotypes and pregnancy outcomes for all cases were sorted out in detail.The ultrasound findings were primarily classified into categories such as the skeletal system, cardiovascular system, brain anomalies, renal anomalies, chest anomalies, orofacial region, non-structural anomalies, etc.

Follow-up outcomes
The follow-up was mainly carried out through telephone interview using the customized questionnaire after all neonates were delivered in our center.The specific followup contents included pregnancy outcomes (miscarriages or birth), gestational age at delivery, sex, birth weight/ length, ultrasound findings during pregnancy (nervous system, cardiovascular system, craniofacial growth, respiratory system, abdominal abnormalities, urinary system, alimentary system, musculoskeletal system and others), and postnatal health conditions (congenital defects, craniofacial dysmorphisms, skeletal anomalies, developmental details and so on).

Study population
Of 20,884 pregnant women opting for prenatal invasive testing, 15 fetuses were identified with 16p11.2microdeletions and five were diagnosed with 16p11.2microduplications.The total detection rate of 16p11.2CNVs was 0.10% (20/20,884) in prenatal setting.Detailed clinical data and follow-up of cases with 16p11.2CNVs are shown in Fig. 1.Tables 1 and 2 summarize the clinical information for all the cases, mainly including gestational week, indications for prenatal diagnosis, parental phenotypes, CMA results, deleted/duplicated regions, inheritance, and pregnancy outcomes.

Chromosomal anomalies detected by karyotyping
Amniotic fluid cells from all pregnant women were subjected to conventional karyotyping to determine whether there were balanced chromosomal rearrangements or mosaicism undetectable by CMA.Among the 15 identified 16p11.2microdeletions and five 16p11.2microduplications, no karyotypic anomalies were detected.

Prenatal and postnatal follow-up assessment
Of the 15 16p11.2deletions cases, 11 chose to terminate their pregnancies: three (case 1, 6 and 9) were de novo, four (case 2, 4, 7 and 14) were parental inheritance, and four (cases 3, 12, 13 and 15) were unavailable.Among the four cases opting for on-going pregnancy, case 5 carried a de novo 16p11.2BP2-BP3 deletion, and the other three (cases 8, 10 and 11) carried maternally inherited 16p11.2BP4-BP5 deletion.It was noteworthy that the mother of case 10 who presented mild learning disability and bradykinesia continued the pregnancy, and delivered a child with no visible abnormalities at birth.However, given the neonate's young age, regular monitoring is necessary to detect any emerging abnormal symptoms.Among the five cases with 16p11.2BP4-BP5 duplication, four (cases 17-20) chose to continue the pregnancies and gave birth to newborns with no visible abnormalities at birth while one (case 16) was lost to follow up.
We conducted follow-up on all neonates with 16p11.2microdeletions and microduplications after birth,

Discussion
In our study, we retrospectively described 20 prenatal cases referred for prenatal invasive testing who were found to carry recurrent chromosomal 16p11.The incidence of 16p11.2CNVs varies in different populations.In the general population, the prevalence of 16p11.2deletions and duplications is 0.028-0.043%and 0.025-0.08%,respectively.For the individuals with neurodevelopmental disorders, the prevalence of 16p11.2deletions and duplications increases to 0.25-2.9%and 0.15-0.78%,respectively [37,40,41].In addition, ASD is found in approximately 25% of individuals with 16p11.2CNVs and about 1% ASD patients carry 16p11.2CNVs [42].The prevalence of 16p11.2CNVs in prenatal settings is sparsely described.In our study, the prevalence of 16p11.2CNVs was 0.10% in prenatal series, with detection rates of 16p11.2deletions and duplications being 0.07% and 0.03%, respectively.Although rarely reported, several studies have also reported the detection rate of 16p11.2CNVs in the prenatal setting.According to the study of Lin et al. [12], the detection rate of 16p11.2microdeletions in fetuses with abnormal ultrasound findings was approximately 0.5% (12/2262).Liu et al. [3] described 24 fetuses (24/8578, 0.28%) with 16p11.2deletions and 6 fetuses (6/8578, 0.07%) with 16p11.2duplications, with a total detection rate of 0.35%.Liu et al. [13] discovered that the prevalence of 16p11.2deletions was 0.063% (55/86,035) in the prenatal period, which was similar to our study.In the study of Wang et al. [43], 1.63% of fetuses (81/4968) were diagnosed with 16p11.2microdeletions, which was higher than other studies.Based upon the results mentioned above, the prevalence of 16p11.2deletions and 16p11.2duplications in prenatal period was 0.063-1.63%and 0.03-0.07%,respectively.More large-scale studies are needed to further clarify the frequencies of 16p11.2CNVs in fetuses.
As one of the most frequent recurrent CNVs associated with neurodevelopmental disorders, the clinical features of 16p11.2CNVs is characterized by phenotypic diversity and incomplete penetrance.Patients carrying 16p11.2CNVs may exhibit a wide spectrum of clinic manifestations, including ID, ASD, ADHD, epilepsy, language disorders, schizophrenia, obesity, congenital malformations, and cardiovascular anomalies [33,[44][45][46].So far, most of Hence, to provide a better understanding of 16p11.2CNVs in prenatal period, we made a pooled analysis of the fetuses carrying 16p11.2microdeletions and microduplications based on the literature review (Tables 3  and 4).The specific breakpoints were classified into BP2-BP3 and BP4-BP5.The most common ultrasound findings in cases of 16p11.2BP2-BP3 deletion included FGR and single umbilical artery.For 16p11.2 BP4-BP5 deletion, the three top structural malformations were abnormality of the vertebral column or rib, renal anomalies and ventricular/atrial septal defect; the three top non-structural malformations were thickened NT, nasal bone absence or hypoplasia, and fetal ventriculomegaly.It is noteworthy that echogenic bowel, observed in our cases, has not been reported in previous fetuses with 16p11.2BP4-BP5 deletion.For 16p11.2 BP4-BP5 duplication, only polyhydramnios and right aortic arch were recurrent prenatal phenotypes.No recurrent prenatal phenotype was observed in 16p11.2BP2-BP3 duplication.No ultrasound anomalies were observed in our cases of 16p11.2BP4-BP5 duplication.Generally speaking, CNVs at the 16p11.2locus can lead to a range of prenatal symptoms, from normal to abnormal, whether they are microduplications or microdeletions.16p11.2BP4-BP5 deletion could present some typical characteristics during the pregnancy period.For 16p11.2 BP2-BP3 deletion and 16p11.2duplications, more clinical cases need to be accumulated to clarify the prenatal features.Refining the prenatal phenotypes associated with different breakpoints of 16p11.2CNVs enables us to provide more accurate genetic counseling.In addition, we found that the incidence of ultrasound abnormalities in 16p11.2deletions was higher than that in 16p11.2duplications (P < 0.01).The rate of TOP in 16p11.2deletions was also higher than that in 16p11.2duplications (P < 0.01).The final pregnancy outcomes would probably be affected by multiple factors, including CNVs classification, the severity of ultrasound abnormalities, and possible future prognosis.
In our study, chromosome 16p11.2BP4-BP5 CNVs were detected in 15 cases, including 10 16p11.2deletions (cases 6-15) and five 16p11.2duplications (cases 16 to 20).According to the DECIPHER database, 23 OMIM genes were located in the overlapping region, among which five were morbid genes associated with diseases (Fig. 2A).TBX6 gene encodes a transcription factor, which is implicated in paraxial mesoderm development and somitogenesis during embryonic development.The haploinsufficiency of TBX6 is supposed to play a critical role in the abnormal phenotypes of the skeleton and kidney.According to the OMIM database, the heterozygous or compound heterozygous mutations in the TBX6 gene would cause spondylocostal dysostosis (SCDO5), characterized by developmental vertebral and rib defects [47].Hemivertebra was observed in our case 13, which might be due to the haploinsufficiency of TBX6.In addition, heterozygous mutations of TBX6 probably lead to genitourinary tract malformations, which might explain the renal agenesis observed in our case 9.It was reported that the increased TBX6 gene dosages could also induce congenital cervical vertebral malformations in humans and mice, but these findings have not been reported in published prenatal cases till now [48].SEZ6L2 encodes a seizure-associated protein localized on the cell surface.It is regarded as a seizure-related gene [46].The haploinsufficiency of SEZ6L2 gene might also be associated with language delay, cognitive impairment, and autism [49].Heterozygous mutations in the KIF22 gene could cause spondyloepimetaphyseal dysplasia with joint laxity (SEMDJL), which is an autosomal-recessive skeletal dysplasia characterized by short stature, generalized joint laxity, slender hands, limb malalignment, and spinal deformity [50,51].ALDOA gene encodes fructose-1,6-bisphosphate aldolase A, and its mutations would cause Glycogen storage disease XII.Altering the ALDOA dosage will perturb energy metabolism at many stages in the brain and affect its development [52].For PRRT2 gene, there is sufficient evidence for haploinsufficiency (HI score:3) recorded in ClinGen database.The PRRT2 mutations would lead to paroxysmal kinesigenic dyskinesia (PKD) and paroxysmal hypnogenic dyskinesia (PHD) in adults and self-limited familial neonatal-infantile epilepsy or infantile convulsion and choreoathetosis (ICCA) in infants [53].The TLCD3B gene encodes the most highly expressed ceramide synthase in human retina.Its homozygous mutations would result in cone-rod dystrophy-22 (CORD22), which would lead to the loss of central vision due to the cone photoreceptor degeneration [54].Some clinical evidence of other genes located in this region were described in other research.The haploinsufficiency of the HIRIP3 gene was probably associated with cardiac arterial valve malformations [55].MAPK3 could regulate the neurodevelopment in ASD and schizophrenia [13].With current knowledge, some evidence shows that some OMIM genes might be responsible for the abnormal phenotypes in the prenatal setting.Further studies are still needed to improve the understanding of the functions of the genes in this region.
Chromosome 16p11.2BP2-BP3 deletions were identified in five cases (cases 1-5).According to the DECI-PHER database, nine OMIM genes were located in the overlapping region, four of which are morbid genes associated with diseases (Fig. 2A).The ATP2A1 gene encodes the fast-twitch skeletal muscle sarcoplasmic reticulum Ca(2+) ATPase; homozygous or compound heterozygous mutations in this gene cause Brody myopathy, characterized by exercise-induced impairment of muscle relaxation and stiffness [56].The haploinsufficiency of ATP2A1 may be associated with diaphragm malformations [17].ATP2A1 is also related to cardiac abnormalities [10].Homozygous or compound heterozygous mutations in the TUFM gene cause combined oxidative phosphorylation deficiency 4, characterized by severe early-onset lactic acidosis and progressive fatal infantile encephalopathy [57].The CD19 and LAT genes are associated with immunodeficiency [10].In addition, the disease-causing gene SH2B1 encodes the Src homology 2B adaptor protein 1, which is involved in leptin and insulin signalling.There is little evidence for haploinsufficiency (HI score:1) recorded in ClinGen database for SH2B1 gene, including developmental delay, severe obesity, hyperphagia and insulin resistance [9,58].Since the prenatal phenotypes of 16p11.2BP2-BP3 CNVs were limited and untypical, additional clinical reports should be provided to further clarify the prenatal genotype-phenotype correlation.
Our study has some limitations.First, the subjects were collected in one single center, and the sample size is relatively small.Multi-center collaboration should be adopted to enlarge the sample size to establish a clearer correlation between 16p11.2deletions/duplications and prenatal phenotypes in the future.Second, not all fetuses carrying 16p11.2CNVs would exhibit abnormal ultrasound findings during the pregnancy period.Long-term follow-up, including postnatal evaluation, should be carried out regularly for those fetuses after birth.In addition, some single gene mutations detected using whole exome sequencing might also be the genetic etiology of the ultrasound anomalies, not just the pathogenic CNVs.Considering the incomplete penetrance and variable expressivity of 16p11.2CNVs, further investigation is needed to establish a more detailed prenatal phenotypegenotype correlation.

Conclusion
In this study, we delineated the clinical data and molecular findings in 20 prenatal cases carrying 16p11.2deletions/duplications.For the first time, we summarized the prenatal features of 16p11.2CNVs in diverse breakpoints based upon the published literature.16p11.2CNVs can manifest diverse prenatal phenotypes, ranging from normal to abnormal.For 16p11.2 BP4-BP5 deletion, the abnormality of the vertebral column or rib and thickened NT were the most common structural and non-structural abnormalities, respectively.In addition, echogenic bowel observed in our study might also be correlated with 16p11.2BP4-BP5 deletion.16p11.2BP2-BP3 deletion was closely correlated with FGR and single umbilical artery.For 16p11.2 duplication, more clinic reports should be accumulated to clarify the prenatal manifestations.Since some abnormal phenotypes associated with 16p11.2CNVs may not be recognizable in neonates, long-term follow-up is necessary regardless of whether they exhibit abnormal intrauterine phenotypic features or not.

Fig. 1
Fig.1The flow chart of the study.CMA: chromosome microarray analysis; CNVs: copy number variations; TOP: termination of pregnancy

Fig. 2
Fig. 2 Scale representation of the deleted/duplicated region in the 16p11.2region (https://decipher.sanger.ac.uk/): (A) Location of genes and genomic syndromes in the 16p11.2locus; (B) Deleted fragments in the present cases; (C) Duplicated fragments in the present cases.Genomic parameters are from GRCh38/hg38

Table 2
Summary of clinical and molecular findings of fetuses presenting 16p11.2microduplications detected by CMA CMA: chromosomal microarray analysis, n.a.: not available, P: pathogenic, AMA: advanced maternal age, mat: maternal, pat: paternal including congenital defects, craniofacial dysmorphisms, skeletal anomalies, as well as other developmental details.Overall, no visible abnormalities have been observed for these cases until this writing.Given the young age of all subjects, some abnormal clinical phenotypes may appear with increasing age.Neurological development assessment should be conducted, and long-term followup should be ensured until adulthood, with a particular focus on neurodevelopmental and behavioral disorders.

Table 3
The pooled data from all fetuses presenting 16p11.2microdeletion

Table 4
The pooled data from all fetuses presenting 16p11.2microduplication